Large stokes shift dyes

ABSTRACT

Provided herein are heptamethine cyanine dyes having a large Stokes shift, and the salts and conjugates thereof. Also provided are methods of using and making such large Stokes shift dyes as fluorescence resonance energy transfer (FRET) acceptors or donors.

RELATED APPLICATIONS

This application is a Division of U.S. Non Provisional application Ser.No. 14/566,166, filed Dec. 10, 2014, which is a Continuation of U.S.Non-Provisional application Ser. No. 13/148,913, filed Aug. 10, 2011,which is a U.S. National Stage Application of PCT application no.PCT/US10/23793, filed Feb. 10, 2010, which claims priority to U.S.Provisional Application No. 61/151,757, filed Feb. 11, 2009, whichdisclosures are herein incorporated by reference in their entirety.

TECHNICAL FIELD

The disclosure relates to fluorescent dye molecules. More particularly,described herein are fluorescent heptamethine cyanine dyes having a longStokes shift and the uses of such dyes in fluorescence resonance energytransfer (FRET), multicolor imaging, flow cytometry, and biomoleculelabeling.

BACKGROUND ART

Heptamethine cyanine dyes have attracted considerable interest becauseof their ability to fluoresce in the near infrared (NIR) region.However, the utility of such dyes in bioassays and in vivo applicationshas been limited because many dyes of this class exhibitphoto-instability, poor water solubility, and a relatively small Stokesshift, typically less than about 25 nm. The Stokes shift is thedifference (in wavelength or frequency units) between the peakabsorption and emission spectra for the same electronic transition. Dyeshaving a small Stoke shifts frequently lack sensitivity due toself-quenching and interference by excitation and scattered light.

Recently, there have been reports of heptamethine cyanine dyes thatexhibit increased Stokes shift properties. For example, Pham et al.described the preparation and properties of a near infrared dye,4-sulfonir, having a large Stokes shift. See Pham et al., Chem. Commun.(2008), 1895-97. Other enamine-containing dyes have been disclosed byPeng et al. to have a large Stokes shift and strong fluorescence. SeePeng et al., J. Am. Chem. Soc. (2005), 127:4170-71. Related dyes and dyeconjugates have been reported by Wang et al. as diagnostic contrastagents. See Wang et al., U.S. Publication No. 20080206886 (publishedAug. 28, 2008).

In spite of these advances, there remains a need to identifyheptamethine cyanine dyes that are both photostable and chemicallystable, and have an increased Stokes shifts, as well as a high quantumyield and molar extinction coefficient. Dyes exhibiting good watersolubility suitable for use in bioassays are particularly desirable.

SUMMARY

Provided herein are heptamethine cyanine dyes, and the salts andconjugates thereof, having a large Stokes shift (i.e., the differencebetween the wavelength at which the dye has maximum absorbance and thewavelength at which the dye has maximum emission), that allows thefluorescent emission to be readily distinguished from the light sourceused to excite the dye. Such large Stokes shift (LSS) dyes preferablyhave a high quantum yield and a large extinction coefficient. Thisdisclosure also relates to using LSS dyes in biomolecule labeling andmethods of using LSS dyes as fluorescence resonance energy transfer(FRET) acceptors or donors.

In one aspect, a compound of Formula I or II is provided:

or a salt or conjugate thereof;

wherein each of R¹ and R² can independently be H, halo, hydroxyl, amino,C₁₋₆ alkylamino, C₂₋₁₂ dialkylamino, C₁₋₆perfluoroalkyl, C₁₋₆ alkyl,C₁₋₆ alkoxy, SO₃H or COOH;

R³ and R⁴ can independently be a (CH₂)_(m)SO₃H, (CH₂)_(n)COOH or C₁₋₆alkyl which can be optionally substituted one or more times by a halo,hydroxyl, C₁₋₆ alkyl, or amino, wherein m and n can independently beintegers from 1 to 6;

R⁵ and R⁶ can independently be a H, C₁₋₆ carbonyl, or C₁₋₆ alkyl whichcan be optionally substituted one or more times by an amino, maleimide,thiol, isocyanate, isothiocyanate, disulfide, alkynyl, azidoyl ortrialkoxysilane;

R⁷ and R⁸ can independently be H, C₁₋₄ alkyl, or phenyl;

X and Y can independently be CR⁹ ₂, NR¹⁹, O, S, or Se; and

each of R⁹ and R¹⁰ can independently be C₁₋₄ alkyl.

In another aspect, a compound of Formula I or II, is provided:

or a salt or conjugate thereof;

wherein each of R¹ and R² can independently be H, halo, hydroxyl, amino,C₁₋₆ alkylamino, C₂₋₁₂ dialkylamino, C₁₋₆ perfluoroalkyl, C₁₋₆ alkyl,C₁₋₆ alkoxy, SO₃H or COOH;

R³ and R⁴ can independently be a (CH₂)_(m)SO₃H, (CH₂)_(n)COOH or C₁₋₆alkyl which can be optionally substituted one or more times by a halo,hydroxyl, C₁₋₆ alkyl, or amino, wherein

m and n can independently be integers from 1 to 6;

R⁵ and R⁶ can be taken together with the nitrogen atom to which they areattached to form an optionally substituted 5- or 6-membered azacyclicring, optionally containing an additional heteroatom selected from N, Oand S as a ring member;

R⁷ and R⁸ can independently be H, C₁₋₄ alkyl, or phenyl;

X and Y can independently be CR⁹ ₂, NR¹⁰, O, S or Se; and

each R⁹ and R¹⁰ can independently be C₁₋₄ alkyl.

In a further aspect, a compound of Formula III, is provided:

or a salt or conjugate thereof;

wherein each of R¹ and R² can independently be H, halo, hydroxyl, amino,C₁₋₆ alkylamino, C₂₋₁₂ dialkylamino, C₁₋₆perfluoroalkyl, C₁₋₆ alkyl,C₁₋₆ alkoxy, SO₃H or COOH;

R³ and R⁴ can independently be a (CH₂)_(m)SO₃H, (CH₂)_(n)COOH or C₁₋₆alkyl which can be optionally substituted one or more times by a halo,hydroxyl, C₁₋₆ alkyl, or amino, wherein

m and n can independently be integers from 1 to 6;

R⁵ and R⁶ can independently be a H, C₁₋₆ carbonyl, or C₁₋₆ alkyl whichcan be optionally substituted one or more times by an amino, maleimide,thiol, isocyanate, isothiocyanate, disulfide, alkynyl, azidoyl ortrialkoxysilane;

R⁷ and R⁸ can independently be H, C₁₋₄ alkyl, or phenyl; and

each R⁹ can independently be C₁₋₄ alkyl.

In another aspect, a compound of Formula III, is provided:

or a salt or conjugate thereof;

wherein each of R¹ and R² can independently be H, halo, hydroxyl, amino,C₁₋₆ alkylamino, C₂₋₁₂ dialkylamino, C₁₋₆perfluoroalkyl, C₁₋₆ alkyl,C₁₋₆ alkoxy, SO₃H or COOH;

R³ and R⁴ can independently be a (CH₂)_(m)SO₃H, (CH₂)_(n)COOH or C₁₋₆alkyl which can be optionally substituted one or more times by a halo,hydroxyl, C₁₋₆ alkyl, or amino, wherein

m and n can independently be integers from 1 to 6;

R⁵ and R⁶ cab be taken together with the nitrogen atom to which they areattached to form an optionally substituted 5- or 6-membered azacyclicring, optionally containing an additional heteroatom selected from N, Oand S as a ring member;

R⁷ and R⁸ can independently be H, C₁₋₄ alkyl, or phenyl; and each R⁹ canindependently be C₁₋₄ alkyl.

In yet another aspect, a compound selected from:

or a salt or conjugate thereof, is provided.

In another aspect, a fluorescing molecular complex, is provided,comprising:

a donor dye capable of absorbing light at a first wavelength andemitting excitation energy in response; and

an acceptor dye capable of absorbing the excitation energy emitted bythe donor dye and fluorescing at a second wavelength in response;

wherein either said donor dye or said acceptor dye has the structure ofFormula I, II, III, IV, V, VI or VII or a salt or conjugate thereof, asfurther described herein.

In one embodiment, a fluorescing molecular complex, is provided,comprising:

a donor dye capable of absorbing light at a first wavelength andemitting excitation energy in response; and

an acceptor dye capable of absorbing the excitation energy emitted bythe donor dye and fluorescing at a second wavelength in response;

wherein either said donor dye or said acceptor dye has the structure ofFormula III:

or a salt or conjugate thereof;

wherein each of R¹ and R² can independently be H, halo, hydroxyl, amino,C₁₋₆ alkylamino, C₂₋₁₂ dialkylamino, C₁₋₆perfluoroalkyl, C₁₋₆ alkyl,C₁₋₆ alkoxy, SO₃H or COOH;

R³ and R⁴ can independently be a (CH₂)_(m)SO₃H, (CH₂)_(n)COOH or C₁₋₆alkyl which can be optionally substituted one or more times by a halo,hydroxyl, C₁₋₆ alkyl, or amino, wherein

m and n can independently be integers from 1 to 6;

R⁵ and R⁶ can independently be a H, C₁₋₆ carbonyl, or C₁₋₆ alkyl whichcan be optionally substituted one or more times by an amino, maleimide,thiol, isocyanate, isothiocyanate, disulfide, alkynyl, azidoyl ortrialkoxysilane;

R⁷ and R⁸ can independently be H, C₁₋₄ alkyl, or phenyl; and

each R⁹ can independently be a C₁₋₄ alkyl.

In another embodiment, a fluorescing molecular complex, is provided,comprising:

a donor dye capable of absorbing light at a first wavelength andemitting excitation energy in response; and

an acceptor dye capable of absorbing the excitation energy emitted bythe donor dye and fluorescing at a second wavelength in response;

wherein either said donor dye or said acceptor dye has the structure ofFormula III:

or a salt or conjugate thereof;

wherein each of R¹ and R² can independently be H, halo, hydroxyl, amino,C₁₋₆ alkylamino, C₂₋₁₂ dialkylamino, C₁₋₆ perfluoroalkyl, C₁₋₆ alkyl,C₁₋₆ alkoxy, SO₃H or COOH;

R³ and R⁴ can independently be a (CH₂)_(m)SO₃H, (CH₂)_(n)COOH or C₁₋₆alkyl which can be optionally substituted one or more times by a halo,hydroxyl, C₁₋₆ alkyl, or amino, wherein

m and n can independently be integers from 1 to 6;

R⁵ and R⁶ can be taken together with the nitrogen atom to which they areattached to form an optionally substituted 5- or 6-membered azacyclicring, optionally containing an additional heteroatom selected from N, Oand S as a ring member;

R⁷ and R⁸ can independently be H, C₁₋₄ alkyl, or phenyl; and

each R⁹ can independently be C₁₋₄ alkyl.

In further embodiments, a compound of Formula IV, V or VI:

or a salt or conjugate thereof, is provided;

wherein each of R¹ and R² can independently be H, halo, hydroxyl, amino,C₁₋₆ alkylamino, C₂₋₁₂ dialkylamino, C₁₋₆ perfluoroalkyl, C₁₋₆ alkyl,C₁₋₆ alkoxy, SO₃H or COOH;

R³ and R⁴ can independently be a (CH₂)_(m)SO₃H, (CH₂)_(n)COOH or C₁₋₆alkyl which can be optionally substituted one or more times by a halo,hydroxyl, C₁₋₆ alkyl, or amino, wherein

m and n can independently be integers from 1 to 6;

R⁵ and R⁶ can independently be a H, C₁₋₆ carbonyl, or C₁₋₆ alkyl whichcan be optionally substituted one or more times by an amino, maleimide,thiol, isocyanate, isothiocyanate, disulfide, alkynyl, azidoyl ortrialkoxysilane;

R⁷ and R⁸ can independently be H, C₁₋₄ alkyl, or phenyl;

X and Y, where present, can independently be CR⁹ ₂, NR¹⁰, O, S or Se;and

each R⁹ and R¹⁹ can independently be C₁₋₄ alkyl.

In still further embodiments, a compound of Formula IV, V, VI, or VII:

or a salt or conjugate thereof, is provided;

wherein each of R¹ and R² can independently be H, halo, hydroxyl, amino,C₁₋₆ alkylamino, C₂₋₁₂ dialkylamino, C₁₋₆ perfluoroalkyl, C₁₋₆ alkyl,C₁₋₆ alkoxy, SO₃H or COOH;

R³ and R⁴ can independently be a (CH₂)_(m)SO₃H, (CH₂)_(n)COOH or C₁₋₆alkyl which can be optionally substituted one or more times by a halo,hydroxyl, C₁₋₆ alkyl, or amino, wherein

m and n can independently be integers from 1 to 6;

R⁵ and R⁶ can be taken together with the nitrogen atom to which they areattached to form an optionally substituted 5- or 6-membered azacyclicring, optionally containing an additional heteroatom selected from N, Oand S as a ring member;

R⁷ and R⁸ can independently be H, C₁₋₄ alkyl, or phenyl;

X and Y, where present, can independently be CR⁹ ₂, NR¹⁰, O, S or Se;and

each R⁹ and R¹⁹ can independently be C₁₋₄ alkyl.

It should be understood that compounds of Formulae I, II, III, IV, V,VI, and VII are capable of forming tautomers, and that all tautomericforms of the compounds disclosed herein are included within the scope ofwhat is described herein.

Compounds described herein are frequently prepared in the form of salts,and salt forms are included within the scope of those embodiments. Theformation of salts, and the number and nature of counterions present,will depend on, e.g., the pH of the solution, the reagents used, and thefunctional groups present in the molecule.

In addition, the various embodiments of the compounds described hereinmay be conjugated to other moieties, including biomolecules, affinitymolecules, ligands, inorganic substrates, and the like, as furtherdescribed herein. Such conjugates are also included within the scope ofthe embodiments described herein.

The LSS dyes described herein may be used in a variety of applications,including bioassays and in vivo applications. In one aspect, theembodiments provide methods of using the compounds described herein, orsalts or conjugates thereof, as FRET donors, or preferably, as FRETacceptors. In particular, certain compounds described herein absorbstrongly at around 605 nm, and emit strongly around 750-800 nm, allowingthem to serve as the reddest (i.e., emit at the upper wavelength rangeof visible light) acceptors for FRET applications.

In another aspect, methods of using the various embodiments of thecompounds described herein for multicolor imaging are provided. In afurther aspect, methods of using the various embodiments of thecompounds described herein for flow cytometry, are further provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the excitation and emission spectra of LSS-Cy dye.

FIG. 2A shows the absorption spectra of the LSS-Cy dye deoxyguanosinetetraphosphate (dG4P) conjugate, LSS-Cy-dG4P; FIG. 2B shows the emissionspectra of LSS-Cy-dG4P.

FIG. 3 shows the excitation and emission spectra of LSS-BA dye.

FIG. 4A shows the absorption spectra of the LSS-BA dye dG4P conjugate,LSS-BA-dG4P; FIG. 4B shows the emission spectra of LSS-BA-dG4P.

DETAILED DESCRIPTION

The embodiments described herein may be understood more readily byreference to the following detailed description of the embodiments andthe Examples included herein. It should be understood that theterminology used herein is for the purpose of describing specificembodiments only and is not intended to be limiting. Furthermore, in thefollowing detailed description of the embodiments, numerous specificdetails are set forth in order to provide a thorough understanding ofthem.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art.

As used herein, “a” or “an” means “at least one” or “one or more.”

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where the event occurs and instances where it does not.

As used herein, “about” means that the numerical value is approximateand small variations would not significantly affect the practice of theembodiments described herein and remain within the scope of theembodiments. Where a numerical limitation is used, unless indicatedotherwise by the context, “about” means the numerical value can vary by±10% and remain within the scope of the embodiments.

As used herein, “water soluble” means the item is soluble or suspendablein an aqueous-based solution, such as in water or water-based solutionsor buffer solutions, including those used in biological or moleculardetection systems as known by those skilled in the art.

The Stokes shift of a dye refers to the difference between thewavelength at which the dye has maximum absorbance and the wavelength atwhich the dye has maximum emission. As used to herein, a “large Stokesshift” (LSS) dye refers to a dye in which the Stokes shift is greaterthan about 30 nm, sometimes greater than about 50 nm, and preferablygreater than about 70 nm. The various embodiments of the compoundsdescribed herein typically have a Stokes shift ranging from about 30 nmto about 250 nm, sometimes from about 50 nm to about 225 nm, preferablyfrom about 70 nm to about 200 nm.

Moreover, it should be appreciated that any of the compounds describedherein can be part of a substantially pure compound compositiontypically containing between about 70% to about 100% of a particularcompound, sometimes containing between about 80% to about 95% of aparticular compound, preferably containing between about 90% to about95% of a particular compound.

The terms “molar extinction coefficient” or “molar absorptivity” (i.e.,epsilon, E, in units of M⁻¹cm⁻¹) relate to the absorption efficiency, orhow strongly a substance absorbs light at a given wavelength per molarconcentration.

The quantum yield of a radiation-induced process is the number of timesthat a defined event, such as emission, occurs per photon absorbed. Asused herein, the term “quantum yield” is a measure of the emissionefficiency.

As used herein, the terms “alkyl,” “alkenyl” and “alkynyl” includestraight-chain, branched-chain and cyclic monovalent hydrocarbylradicals, and combinations thereof, which contain only C and H when theyare unsubstituted. Examples include, but are not limited to, methyl,ethyl, isobutyl, cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butynyl,and the like. The total number of carbon atoms in each such group issometimes described herein, e.g., when the group can contain up to tencarbon atoms it may be described as 1-10C or as C₁-C₁₀ or as C₁₋₁₀. Whenheteroatoms (such as N, O and S) are allowed to replace carbon atoms ofan alkyl, alkenyl or alkynyl group, as in heteroalkyl groups, forexample, the numbers describing the group, though still written as e.g.C₁-C₆, represent the sum of the number of carbon atoms in the group plusthe number of such heteroatoms that are included as replacements forcarbon atoms in the ring or chain being described.

Typically, the alkyl, alkenyl and alkynyl substituents of theembodiments described herein contain, but are not limited to, 1-10C(alkyl) or 2-10C (alkenyl or alkynyl). Sometimes they contain 1-8C(alkyl) or 2-8C (alkenyl or alkynyl). Preferably, they contain 1-6C(alkyl) or 2-6C (alkenyl or alkynyl). Sometimes they contain 1-4C(alkyl) or 2-4C (alkenyl or alkynyl). A single group can include morethan one type of multiple bond, or more than one multiple bond; suchgroups are included within the definition of the term “alkenyl” whenthey contain at least one carbon-carbon double bond, and they areincluded within the term “alkynyl” when they contain at least onecarbon-carbon triple bond.

Alkyl, alkenyl and alkynyl groups are often substituted to the extentthat such substitution can chemically occur. Typical examples ofsubstituents can include, but are not limited to, halo, acyl,heteroacyl, carboxylic acid, sulfonic acid, primary or secondary amine,thiol, hydroxyl, or an activated derivative thereof, or a protected formof one of these. Alkyl, alkenyl and alkynyl groups can also besubstituted by C₁-C₈ acyl, C₂-C₈ heteroacyl, C₆-C₁₀ aryl or C₅-C₁₀heteroaryl, each of which can be substituted by the substituents thatare appropriate for the particular group.

As used herein, “acyl” encompasses groups comprising an alkyl, alkenyl,alkynyl, aryl or arylalkyl radical attached at one of the two availablevalence positions of a carbonyl carbon atom, e.g., —C(═O)R where R canbe an alkyl, alkenyl, alkynyl, aryl, or arylalkyl group, and heteroacylrefers to the corresponding groups wherein at least one carbon otherthan the carbonyl carbon has been replaced by a heteroatom chosen fromN, O and S. Thus heteroacyl includes, for example, —C(═O)OR and—C(═O)NR₂ as well as —C(═O)-heteroaryl, where each R is independently H,or C1-C8 alkyl.

“Aromatic” moiety or “aryl” moiety refers to a monocyclic or fusedbicyclic moiety having the well-known characteristics of aromaticity;examples include, but are not limited to, phenyl and naphthyl.Similarly, “heteroaromatic” and “heteroaryl” refer to such monocyclic orfused bicyclic ring systems which contain as ring members one or moreheteroatoms (such as O, S and N). The inclusion of a heteroatom permitsaromaticity in 5-membered rings as well as 6-membered rings. Typicalheteroaromatic systems include, but are not limited to, monocyclic C5-C6aromatic groups such as pyridyl, pyrimidyl, pyrazinyl, thienyl, furanyl,pyrrolyl, pyrazolyl, thiazolyl, oxazolyl, and imidazolyl, and the fusedbicyclic moieties formed by fusing one of these monocyclic groups with aphenyl ring or with any of the heteroaromatic monocyclic groups to forma C8-C10 bicyclic group such as indolyl, benzimidazolyl, indazolyl,benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl,pyrazolopyridyl, quinazolinyl, quinoxalinyl, cinnolinyl, and the like.Preferably aryl groups contain 6-10 ring members, and heteroaryl groupscontain 5-10 ring members.

Aryl and heteroaryl moieties may be substituted with a variety ofsubstituents including C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C5-C12aryl, C1-C8 acyl, and heteroforms of these, each of which can itself befurther substituted; other substituents for aryl and heteroaryl moietiescan include halo, OR, NR₂, SR, SO₂R, SO₂NR₂, NRSO₂R, NRCONR₂, NRCOOR,NRCOR, CN, COOR, CONR₂, OOCR, —C(O)R, and NO₂, wherein each R isindependently H, or C1-C8 alkyl.

In one aspect, a compound of Formula I or II:

or a salt or conjugate thereof, is provided.

In compounds of Formula I and Formula II, R¹ and R² can independently beH, halo, hydroxyl, amino, C₁₋₆ alkylamino, C₂₋₁₂ dialkylamino, C₁₋₆perfluoroalkyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, SO₃H or COOH. In someembodiments, R¹ and R² are different. In other embodiments, R¹ and R²are the same. In some such embodiments, each of R¹ and R² is SO₃H.

In compounds of Formula I and Formula II, R³ and R⁴ can independently bea (CH₂)_(m)SO₃H, (CH₂)_(n)COOH or C₁₋₆ alkyl (which can be optionallysubstituted one or more times by a halo, hydroxyl, C₁₋₆ alkyl, oramino), wherein m and n are independently integers from 1 to 6. Incertain embodiments, each of m and n is an integer from 2 to 4. In someembodiments, R³ and R⁴ are different. In other embodiments, R³ and R⁴are the same. In certain embodiments, each of R³ and R⁴ is(CH₂)_(m)SO₃H. In some such embodiments, m is an integer from 2 to 4; inspecific embodiments, m is 3.

In some embodiments of Formula I and Formula II, R⁵ and R⁶ canindependently be H, C₁₋₆ carbonyl, or optionally substituted C₁₋₆ alkyl.

Optional substituents when present on the C₁₋₆ alkyl groups at R⁵ and R⁶include for example, halo, acyl, heteroacyl, carboxylic acid, primary orsecondary amine, thiol, hydroxyl, maleimide, isocyanate, isothiocyanate,disulfide, alkynyl, azidoyl, trialkoxysilane, or an activated derivativethereof. Activated derivatives of carboxylic acids can include, e.g.,activated esters, such as N-hydroxysuccinimide (NHS) esters,hydroxybenzotriazole (HOBT) or 1-hydroxy-7-aza-benzotriazole (HOAt)esters, or mixed anhydrides. Activated amines can include, e.g., N-acylimidazolides, while activated thiols and hydroxyls can include, e.g.,imidazole carbamate and thiocarbamate derivatives, and succinimidylcarbonates and thiocarbonate derivatives, and sulfonate esters andthioesters. Other suitable activated derivatives are known to those ofskill in the art.

In other embodiments of Formula I and Formula II, R⁵ and R⁶ are takentogether with the nitrogen atom to which they are attached to form anoptionally substituted 5- or 6-membered azacyclic ring substituted,optionally containing an additional heteroatom selected from N, O and Sas a ring member.

Optional substituents when present on the azacyclic ring formed by R⁵and R⁶ can include, for example, halo, acyl, heteroacyl, carboxylicacid, primary or secondary amine, thiol, hydroxyl, or C₁₋₆ alkylsubstituted with halo, acyl, heteroacyl, carboxylic acid, primary orsecondary amine, thiol, or hydroxyl, or an activated derivative of anyof the above, where activated derivatives for various functional groupsare the same as those provided above for substiuents on C₁₋₆ alkylgroups at R⁵ and R⁶.

In compounds of Formula I and Formula II, R⁷ and R⁸ can independently beH, C₁₋₄ alkyl, or phenyl. In some embodiments, each of R⁷ and R⁸ is H.In other embodiments, one of R⁷ and R⁸ is H and the other is phenyl ortert-butyl.

In compounds of Formula I and Formula II, X and Y can independently beCR⁹ ₂, Ne, O, S, or Se; where each of R⁹ and R¹⁰ is independently C₁₋₄alkyl. In certain embodiments, each R⁹ and R¹⁹ are preferably methyl.

In some embodiments of Formula I and Formula II, X and Y are different.In other embodiments, X and Y are the same. In specific embodiments,each of X and Y is CR⁹ ₂, where each R⁹ is independently C₁₋₄ alkyl. Insome such embodiments, each R⁹ is methyl.

In certain preferred embodiments of Formula I and Formula II, R¹ and R²are the same, R³ and R⁴ are the same, and X and Y are the same.

In another aspect, compound of Formula III:

III

or a salt or conjugate thereof, is provided.

In compounds of Formula III, R¹ and R² can independently be H, halo,hydroxyl, amino, C₁₋₆ alkylamino, C₂₋₁₂ dialkylamino,C₁₋₆perfluoroalkyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, SO₃H or COOH. In someembodiments, R¹ and R² are different. In other embodiments, R¹ and R²are the same. In some such embodiments, each of R¹ and R² is SO₃H.

In compounds of Formula III, R³ and R⁴ can independently be a(CH₂)_(m)SO₃H, (CH₂)_(n)COOH or C₁₋₆ alkyl (which can be optionallysubstituted one or more times by a halo, hydroxyl, C₁₋₆ alkyl, oramino), wherein m and n are independently integers from 1 to 6. Incertain embodiments, each of m and n is an integer from 2 to 4. In someembodiments, R³ and R⁴ are different. In other embodiments, R³ and R⁴are the same. In certain embodiments, each of R³ and R⁴ is(CH₂)_(m)SO₃H. In some such embodiments, m is an integer from 2 to 4; inspecific embodiments, m is 3.

In some embodiments of Formula III, R⁵ can be H, or optionallysubstituted C₁₋₆ alkyl; and R⁶ can be C₁₋₆ carbonyl, or optionallysubstituted C₁₋₆ alkyl. In other embodiments of Formula III, R⁵ and R⁶can be taken together with the nitrogen atom to which they are attachedto form an optionally substituted 5- or 6-membered azacyclic ringsubstituted, optionally containing an additional heteroatom selectedfrom N, O and S as a ring member.

Optional substituents when present on R⁵, R⁶, or on the azacyclic ringformed by R⁵ and R⁶, are the same as those provided herein for compoundsFormula I and Formula II.

In compounds of Formula III, R⁷ and R⁸ can independently be H, C₁₋₄alkyl, or phenyl. In some embodiments, each of R⁷ and R⁸ is H. In otherembodiments, one of R⁷ and R⁸ is H and the other is phenyl ortert-butyl.

In compounds of Formula III, each R⁹ can independently be C₁₋₄ alkyl. Incertain embodiments, each R⁹ is the same; preferably, each R⁹ is methyl.

In certain preferred embodiments of Formula III, R¹ and R² are the same,R³ and R⁴ are the same, and each R⁹ is the same. In some suchembodiments, each of R⁷ and R⁸ is H.

In further embodiments, a compound of Formula IV, V, VI, or VII:

or a salt or conjugate thereof, is provided.

The same groups described herein for X, Y, R¹, R², R³, R⁴, R⁵, R⁶, R⁷,R⁸, R⁹, and R¹⁰ for compounds of Formula I, II and III are also usefulfor compounds of Formula IV, V, VI and VII.

The compounds described above, or the salts and conjugates thereof, havea large Stokes shift which is greater than about 30 nm, sometimesgreater than about 50 nm, and preferably greater than about 70 nm. Insome embodiments, the compounds, salts and conjugates thereof have aStokes shift from about 30 nm to about 250 nm. In other embodiments, theStokes shift is from about 50 nm to about 225 nm. Preferably, the Stokesshift is from about 70 nm to about 200 nm.

Without wishing to be bound by theory, it is believed that the presenceof the enamine functionality on the central cyclohexenyl ringcontributes significantly to the absorption and emission spectra of theembodiments of the dyes described herein. The enamine nitrogen atom issubstituted with the groups R⁵ and R⁶, which can be substituted with afunctional group suitable for conjugation to another moiety, as furtherdescribed herein.

The compounds described herein, or the salts or conjugates thereof, aretypically soluble in water or aqueous solutions or buffers typicallyused in biological systems and assays.

The compounds described herein are particularly useful in applicationsrelated to fluorescence resonance energy transfer (FRET). FRET is adistance-dependent interaction between the electronic excited states oftwo dye molecules in which excitation energy is transferred from a donormolecule to an acceptor molecule. The efficiency of FRET is dependent onthe inverse sixth power of the separation distance between the donormolecule and acceptor molecule (i.e., intermolecular separation), makingit useful over distances comparable with the dimensions of biologicalmacromolecules. Thus, FRET is an important technique for investigating avariety of biological phenomena that produce changes in molecularproximity The embodiments of the compounds, or the salts or conjugatesthereof, may function as either a FRET donor or a FRET acceptor. Incertain embodiments, they are FRET acceptors.

In one aspect, a fluorescing molecular complex, is provided, comprising:a donor dye capable of absorbing light at a first wavelength andemitting excitation energy in response; and

an acceptor dye capable of absorbing the excitation energy emitted bythe donor dye and fluorescing at a second wavelength in response;

wherein either said donor dye or said acceptor dye has the structure ofFormula I, II, III, IV, V, VI, or VII or a salt or conjugate thereof, asfurther described herein.

In specific embodiments, the donor dye or the acceptor dye has thestructure of Formula III, or a salt or conjugate thereof.

In some embodiments, the acceptor dye has the structure of Formula III,or a salt or conjugate thereof. In some such embodiments, the acceptordye has an excitation wavelength of between about 400 nm and about 900nm. In further embodiments, the acceptor dye has an emission wavelengththat is at least 30 nm, sometimes 50 nm, and preferably 70 nm greaterthan the excitation wavelength. In some embodiments, the acceptor dyehas a Stokes shift of about 30 nm to about 250 nm, sometimes about 50 nmto about 225 nm, preferably about 70 nm to about 200 nm.

In further embodiments, the donor dye is a suitably functionalizedquantum dot (QD) nanocrystal or a conjugate thereof, and the acceptordye is one of the compounds described herein, or a salt or conjugatethereof. In some such embodiments, the acceptor dye is a compound ofFormula III, or a salt or conjugate thereof.

In some embodiments, the acceptor dye is LSS-Cy or LSS-BA, or a salt orconjugate thereof. In some such embodiments, the acceptor dye is aconjugate of LSS-Cy or LSS-BA, or a salt thereof. In specificembodiments, the acceptor dye is LSS-Cy or LSS-BA conjugated to anucleoside polyphosphate (e.g., deoxyguanosine tetraphosphate (dG4P),deoxyadenosine tetraphosphate (dA4P), deoxythymidine tetraphosphate(dT4P), deoxycytidine tetraphosphate (dC4p), etc.).

The compounds described herein may be isolated as salts where anionizable acidic or basic group is present. Methods of forming andexchanging salts are well known in the art. For example, salts of acidicgroups may be formed by reaction with organic or inorganic bases, andsalts of basic groups may be formed by reaction with organic orinorganic acids. The formation of salts, including the number and natureof counterions present, will depend on the pH of the solution, thereagents used, and the functional groups present in the molecule.

Examples of inorganic bases include, but are not limited to, thehydroxides, alkoxides, carbonates, acetates and the like, of alkalimetals (e.g., sodium, potassium or lithium) or alkaline earth metals(e.g., calcium), as well as similar salts of aluminum, ammonium, etc.Reaction of carboxylic acids or sulfonic acids with such inorganic basesprovide the corresponding alkali metal salt or alkaline earth metal saltof the carboxylic or sulfonic acid. Such groups may be representedherein as SO₃ ⁻M⁺ or CO₂ ⁻M⁺, where M⁺ represents the metal counterion.

Examples of organic bases that could be used include, but are notlimited to, trimethylamine, triethylamine, pyridine, picoline,ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine,N,N′-dibenzylethylenediamine, etc.

Examples of inorganic acids that could be used include, but are notlimited to, hydrochloric acid, hydrobromic acid, nitric acid, sulfuricacid, phosphoric acid, etc. Examples of organic acids include formicacid, oxalic acid, acetic acid, tartaric acid, methanesulfonic acid,benzenesulfonic acid, malic acid, methanesulfonic acid, benzenesulfonicacid, p-toluenesulfonic acid, etc. Also included are salts with basicamino acids such as arginine, lysine, ornithine, etc., and salts withacidic amino acids such as aspartic acid, glutamic acid, etc.

In some embodiments, the acidic functional groups (i.e., carboxylic orsulfonic acids) that can be present as part of substituent groups R¹,R², R³, R⁴, as well as optional substituents present as part of R⁵, R⁶,or the tether linking them, will be protonated. In some embodiments, aninner salt (or zwitterion) will be formed between the positively chargednitrogen atom of the indolinium ring and the acidic functional grouppresent on the attached substituent, e.g., R³ in the tautomer shown inFormulae I-VI. At sufficiently low pH, all of the acidic groups can beprotonated and the positive charge on the indolinium ring nitrogen canbe counterbalanced by an anionic counterion, such as a halide ion or asulfonate ion, etc. At higher pH, some or all of the acidic groupspresent in the various embodiments of the compounds described hereinwill be deprotonated, and the resulting negative charge(s) balanced byone or more positively charged counterions, such as alkali or alkalineearth metal ions.

Synthesis of LSS Dyes

Exemplary methods for preparing LSS heptamethine cyanine dyes from a2-chloro-cyclohexene precursor are shown in Scheme 1 and Scheme 2.However, it should be understood that preparing the various embodimentsof the LSS heptamethine cyanine dyes disclosed herein may be achievedusing other methods known in the art. Synthetic methods for makingselected compounds disclosed herein are also provided.

Conjugates

The various embodiments of the compounds described herein may be used toprepare a variety of dye conjugates. A dye conjugate can be formed bylinking an embodiment of the LSS heptamethine cyanine dyes describedherein to another moiety under conditions known to those of skill in theart. Frequently, the various embodiments of the LSS heptamethine cyaninedye compounds disclosed herein are conjugated through a functional groupthat is present as an optional substituent on the C₁₋₆ alkyl groups atR⁵ and R⁶, or as part of a substituent on the ring formed when R⁵ and R⁶are taken together. Examples of functional groups useful in suchconjugation reactions include halo, acyl, heteroacyl, carboxylic acid,primary or secondary amine, thiol, hydroxyl, or activated derivativesthereof. When R⁵ and R⁶ are taken together to form a ring, thefunctional group may be directly attached to the ring, or may be asubstituent on a C₁₋₆ alkyl substituent thereon. In addition to stableactivated derivatives, such as the NHS, HOBt and HOAt esters describedpreviously, the functional groups described herein may be transientlyactivated to undergo in situ conjugation, for example by reaction with acoupling reagent such as a carbodiimide or a uronium phosphate reagent(e.g., HBTU).

In some embodiments, the compounds described herein may be directlyconjugated to an appropriately selected moiety, for example, byformation of an amide bond between a carboxylic acid or an aminefunctional group on the dye molecule with a corresponding amine or acidfunctional group on the moiety to be conjugated. In other embodiments,the dye may be coupled to the appropriately selected moiety via anintermediate linker. The linker may be attached to a functional group onthe dye or to the other moiety prior to conjugation. In frequentembodiments, the linker comprises a saturated or unsaturated alkylene orheteroalkylene group. For example, a carboxylic acid on the dye can becoupled to linker molecule comprising an amino-alkanol to form an amidebond, and the hydroxyl group of the alkanol can be further coupled tothe desired moiety.

Examples of suitable moieties to which the compounds described hereinmay form conjugates include, without limitation, nucleotides,nucleosides, nucleic acids, oligonucleotides, deoxyoligonucleotides, DNAfragments, RNA fragments, or a derivatized variant of one of these.Additional examples of conjugatable moieties include antibodies, aminoacids, peptides, and proteins, as well as haptens, antigens, drugs,cells or viruses, carbohydrates, polysaccharides and oligosaccharides,lipids, phospholipids, lipoproteins, lipopolysaccharides, liposomes,polymers, polymeric microparticles, etc.

In some embodiments, the other moiety can be an affinity molecule, suchas an antibody, receptor or enzyme, which specifically recognizes, bindsto or modifies another compound or structure. The dye conjugate, byvirtue of the affinity molecule, can be used to detect, for example, thepresence and/or quantity of biological and chemical compounds,interactions in biological systems, biological processes, alterations inbiological processes, or alterations in the structure of biologicalcompounds. The affinity molecule, when linked to a dye compounddescribed herein, can interact with a biological target that serves asthe second member of the binding pair, in order to detect biologicalprocesses or reactions, or to alter biological molecules or processes.The interaction of the affinity molecule and the biological target mayinvolve specific binding, and can involve covalent, noncovalent,hydrophobic, hydrophilic, electrostatic, van der Waals, magnetic, orother interactions.

The affinity molecule or other moiety associated with a dye can benaturally occurring or artificially synthesized, and can be selected tohave a desired physical, chemical or biological property. Suchproperties can include covalent and noncovalent association with, forexample, signaling molecules, prokaryotic or eukaryotic cells, viruses,subcellular organelles and any other biological compounds. Otherproperties include the ability to affect a biological process, cellcycle, blood coagulation, cell death, transcription, translation, signaltransduction, DNA damage or cleavage, production of radicals, scavengingradicals, the ability to alter the structure of a biological compound,crosslinking, proteolytic cleavage, and radical damage.

In some embodiments, one of the LSS dye compounds described herein maybe conjugated to a molecule or species for detection by means of FRET.In some embodiments, the FRET efficiency in a FRET reaction of thecompounds described herein can be between about 25% to about 100%.

FRET refers to Fluorescence Resonance Energy Transfer (sometimes calledFörster Resonance Energy Transfer) which is the basis of variousfluorescence measuring techniques that allow detection of the closeproximity of two appropriately labeled molecules or species. Asdiscussed above, in FRET, a donor label non-radiatively transfers energyto a second acceptor label. The acceptor may be a fluorophore which maythen emit a photon. Donor-acceptor pairs are typically selected suchthat there is overlap between the emission spectrum of the donor andexcitation spectrum of the acceptor. In some applications, the acceptormay be a quencher.

FRET efficiency depends sharply on donor-acceptor distance r as 1/r⁶.The distance where FRET efficiency is 50% is termed R₀, also known asthe Förster distance. R₀ is unique for each donor-acceptor combinationand may be 5 to 10 nm. In biological applications, FRET can provide anon-off type signal, indicating when the donor and acceptor are within R₀of each other. Additional factors affecting FRET efficiency include thequantum yield of the donor, the extinction coefficient of the acceptor,and the degree of spectral overlap between donor and acceptor. FRETefficiency and signal detection is described in D. W. Piston and G. J.Kremers, Trends Biochem. Sci. 32:407 (2007).

Nanocrystals have been used for FRET detection in biological systems.See, e.g., Willard et al., 2001, Nano. Lett. 1:469; Patolsky F. et al.,2003, J. Am. Chem. Soc. 125:13918; Medintz I. L. et al., 2003, Nat.Mater. 2:630; Zhang C. Y., et al., 2005, Nat. Matter. 4:826.Nanocrystals may be advantageous because their emission may besize-tuned to improve spectral overlap with an acceptor or quencher.Nanocrystals in general have high quantum yield and are less susceptibleto photobleaching than other types of FRET donors.

In some embodiments, the compounds described herein, or a salt orconjugate thereof, functions as a FRET acceptor. In particularembodiments, the corresponding FRET donor is a suitably functionalizedquantum dot (QD) nanocrystal.

In a particular embodiment the FRET donor is a QD605 nanocrystal, or aconjugate thereof. When QD605 is used as the FRET donor, the reddestacceptor channel is limited to the emission window of 750-800 nm. Mostcommercial dyes having emission in this window (e.g., AF750, Cy7, HiLyte750, CF 750, Atto 750, Dy734 and Dy750) have a very small absorptionaround 605 nm (extinction coefficient ˜15,000 M⁻¹cm⁻¹). For example, noFRET signal was observed from QD605 to AF750. By contrast, theLSS-Cy-dG4P and LSS-BA-dG4P dyes absorb strongly around 605 nm, whichmatch the emission of QD605.

In specific embodiments, the LSS-Cy dye or the LSS-BA dye (prepared asdescribed herein) is conjugated to a nucleoside polyphosphate (e.g.,deoxyguanosine tetraphosphate (dG4P), deoxyadenosine tetraphosphate(dA4P), deoxythymidine tetraphosphate (dT4P), deoxycytidinetetraphosphate (dC4P), etc.) as shown in Scheme 3 and Scheme 4,respectively. The carboxylic acid present in the dye molecule wasconverted to an activated NHS ester, and then further reacted with anucleophilic amine and linked to dG4P to provide the conjugates shown.

The following experimental results are offered to illustrate but not tolimit the embodiments described herein.

EXAMPLE 1

The LSS-Cy dye was prepared as shown in Scheme 1 and detailed in thefollowing experimental synthesis workflow.

1. Synthesis of Compound 3:

Compound 1 (i.e., 5-sulfo-1-sulfopropyl-2,3,3-trimethylindoleninium,inner salt, sodium salt) (1 g), compound 2 (i.e.,N-[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]anilinemonohydrochloride) (404 mg) and NaOAc (226 mg) in absolute EtOH (30 mL)were refluxed for 5 hours. After cooling, the precipitates werecollected by filtration and washed with EtOH (30 mL×4) and CHCl3 (30mL×4). The solid was purified by column chromatography on silica gel,eluting with 10% H2O in acetonitrile. Evaporation of the solventafforded compound 3 as a green solid (350 mg).

2. Synthesis of Compound 5 (LSS-Cy Dye):

Compound 3 (i.e.,2-[2-[2-Chloro-3-[2-[5-sulfo-1,3-dihydro-3,3-dimethyl-1-(4-sulfopropyl)-2H-indol-2-ylidene]-ethylidene]-1-cyclohexen-1-yl]-ethenyl]-5-sulfo-3,3-dimethyl-1-(4-sulfopropyl)-3H-indolium)(100 mg), 4 (i.e., isonipecotic acid) (150 mg) and triethylamine (175μL) were dissolved in a mixture of DMF (3 mL) and water (2 mL). Thesolution was heated at about 80-85° C. for about 2 hours. The solventwas evaporated and the residue was purified by column chromatography(C.C.) on silica gel, eluting with 10% water/acetonitrile. The productwas further purified by C.C. on sephadex LH-20, eluting with H2O.Evaporation of the solvent gave ca. 70 mg of compound 5.

The emission spectrum of LSS-Cy was similar to AlexaFluor 750 (AF750).The LSS-Cy dye absorbed strongly at 605 nm (E=66000 M⁻¹cm⁻¹).

Quantum Yield: 0.72 times of that of AF750.

Brightness: ca. 3.5 times of that of AF750 when excited at 605 nm.

Example 2 LSS-Cy-dG4P Conjugate

The LSS-Cy dye was conjugated to dG4P via its NHS ester, as shown inScheme 3 and detailed in the following experimental synthesis workflow.

3. Synthesis of Succinate Ester (SE) Form of Compound 5 (Compound 7)

To a solution of compounds 5 (40 mg) and 6 (27 mg) in drydimethylformamide (DMF) (5 mL) was added triethylamine (100 μL). Thesolution was stirred at room temperature for 1 h. Ethyl ether (ca. 30mL) was slowly added. The precipitate was collected by centrifuge anddried in vacuum (42 mg).

4. Labeling dGP4 with LSS-Cy Dye.

A solution of amino-dG4P (10) (0.5 mg) in DMF-water (2:1, 300 μL) wasmixed with 50 μL of saturated sodium bicarbonate solution. To thissolution was added the LSS-Cy dye SE (7) (2 mg). The solution wasstirred at room temperature until the completion of the reaction (ca. 1hour). The product was purified by column chromatography on sephadexLH-20, eluting with water. The desired fraction was concentrated to ca.300 μL and stored at −20° C.

After conjugation, the absorption spectra of the LSS-Cy-dG4P conjugateshifted to longer wavelength (from 575 nm to 590 nm).

The excitation and emission spectra remained the same, but the quantumyield was increased 1.1 times.

Example 3 LSS-BA Dye

The LSS-BA dye was prepared as shown in Scheme 2 and detailed in thefollowing experimental synthesis workflow.

1. Synthesis of Compound 8 (LSS-BA Dye)

Compound 3 (i.e.,2-[2-[2-Chloro-3-[2-[5-sulfo-1,3-dihydro-3,3-dimethyl-1-(4-sulfopropyl)-2H-indol-2-ylidene]-ethylidene]-1-cyclohexen-1-yl]-ethenyl]-5-sulfo-3,3-dimethyl-1-(4-sulfopropyl)-3H-indolium)(50 mg), β-alanine (52 mg) and triethylamine (76 μL) were dissolved in amixture of DMF (2 mL) and water (1 mL). The solution was heated at about80-85° C. for about 2 hours. The solvent was evaporated and the residuewas purified by column chromatography (C.C.) on silica gel, eluting with10% water/acetonitrile. The product was further purified by C.C. onsephadex LH-20, eluting with H2O. Evaporation of the solvent gave ca. 22mg of compound 8.

The LSS-BA dye absorbed strongly at 601 nm (ε=92000 M⁻¹cm⁻¹), andemitted at 760 nm with a broad peak.

Quantum Yield: 1.55 Times of that of AF750.

Brightness: ca. 11 times of that of AF750 when excited at 605 nm.

Example 4 LSS-BA-dG4P Conjugate

The LSS-BA dye was conjugated to dG4P via its NHS ester, as shown inScheme 4 and detailed in the following experimental synthesis workflow.

2. Synthesis of SE of Compound 8 (Compound 9)

To a solution of compounds 8 (10 mg) and 6 (11 mg) in dry DMF (5 mL) wasadded triethylamine (50 μL). The solution was stirred at roomtemperature for 1 h. Ethyl ether (ca. 30 mL) was slowly added. Theprecipitate was collected by centrifuge and dried in vacuum (14 mg).

3. Labeling dGP4 with LSS-BA Dye.

A solution of amino-dG4P (10) (0.5 mg) in DMF-water (2:1, 300 μL) wasmixed with 50 μL of saturated sodium bicarbonate solution. To thissolution was added the LSS-BA dye SE (9) (2 mg). The solution wasstirred at room temperature until the completion of the reaction (ca. 1hour). The product was purified by column chromatography on sephadexLH-20, eluting with water. The desired fraction was concentrated to ca.300 μL and stored at −20° C.

After conjugation with dG4P, the absorption of the dye conjugate shiftedto longer wavelength (from 600 nm to 617 nm).

The excitation and emission spectra remained the same followingconjugation, but the quantum yield increased by 1.05 times.

By way of example, the amino-dG tetraphosphate (i.e., nucleosidepolyphosphate moiety) that the LSS-Cy and LSS-BA dyes were depictedabove as being conjugated to (in schemes 3 and 4) was prepared as shownin the following experimental synthesis workflow.

Example 5 Amino-dG Tetraphosphate

1. Synthesis of Compound 2.

Compound 1 (678 mg, 2 mmol) was suspended in trimethyl phosphate (5 mL)and cooled to 0° C. POCl3 (280 μL) was added to the stirred mixtureunder argon. The mixture was warmed up and stirred at room temperatureovernight. The reaction was quenched by adding slowly 4 mL of TEABbuffer (1 M) at about 0° C. Triethylamine was added to adjust the pH topH 7.0. The solvent was evaporated and the residue was purified bycolumn chromatography on silica gel, eluting with 10% H20/CH3CN. Afterevaporation of the solvent, the solid was dissolved in water. The pH ofthe solution was adjusted to pH 7 with TEAB buffer (1 M), followed byco-evaporation with methanol. Yield: 400 mg of compound 2.

LSS-BAS, LSS-AL, LSS-SAR, LSS-GL, LSS-ALS, LSS-CY LITE, LSS-SER (Abs.628 nm/em 745 nm, QY=1.36 LSS-BA), LSS-HS (Abs. 597 nm/em. 755 nm,QY=1.0 LSS-BA), LSS-TH (Abs. 637 nm/em. 728 nm, QY=2.0 LSS-BA), LSS-AB(Abs. 623 nm/em. 757 nm, QY=1.1 LSS-BA), LSS-VA (Abs. 628 nm/em. 722 nm,QY=1.1 LSS-BA), LSS-PP (Abs. 612 nm/em. 780 nm), LSS-GL-GL (Abs. 642nm/em. 760 nm), LSS-CER-CY (Abs. 700 nm/em. 770 nm), and LSS-GL-CY dyes(Abs. 635 nm/em. 760 nm) were made by the same method as the LSS-BA andLSS-CY dyes (see Example 1 and Example 3 above).

Unless otherwise specified, all documents referred to herein areincorporated by reference in their entirety.

While certain embodiments have been described above, it will beunderstood that the embodiments are described by way of example only.Those skilled in the art will appreciate that the same can be performedwithin a wide range of equivalent parameters, concentrations, andconditions without departing from the spirit and scope of theembodiments disclosed herein and without undue experimentation.Accordingly, the compositions/compounds, processes and/or methodsdescribed herein should only be limited in light of the claims thatfollow when taken in conjunction with the above description andaccompanying drawings.

1. A compound of Formula I or II:

or a salt or conjugate thereof; wherein R¹ and R², when taken alone, areindependently H, halo, hydroxyl, amino, C₁₋₆ alkylamino, C₂₋₁₂dialkylamino, C₁₋₆ perfluoroalkyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, SO₃H orCOOH; R³ and R⁴, when taken alone, are independently (CH₂)_(m)SO₃H,(CH₂)_(n)COOH or optionally substituted C₁₋₆ alkyl, wherein m and n areindependently integers from 1 to 6; R⁵ and R⁶, when taken alone, areindependently H, C₁₋₆ carbonyl or optionally substituted C₁₋₆ alkyl; R⁷and R⁸ are independently H, C₁₋₄ alkyl, or phenyl; X and Y areindependently CR⁹ ₂, Ne, O or S; and each R⁹ and R¹⁰ is independentlyC₁₋₄ alkyl.
 2. The compound of claim 1, wherein each of X and Y is CR⁹₂.
 3. The compound of claim 1, wherein each of R⁷ and R⁸ is H.
 4. Thecompound of claim 1, wherein at least one of R⁵ and R⁶ is C₁₋₆ alkylsubstituted with a substituent selected from the group consisting ofSO₃H, COOH, OH, NH₂, maleimide, thiol, isocyanate, isothiocyanate,disulfide, alkynyl, azidoyl and trialkoxysilane.
 5. The compound ofclaim 1, wherein at least one of R³ and R⁴ is C₁₋₆ alkyl substitutedwith a substituent selected from the group consisting of halo, hydroxyl,C₁₋₆ alkyl, or amino
 6. The compound of claim 1, wherein each of R¹ andR² is SO₃H.
 7. The compound of claim 1, wherein each of R³ and R⁴ is(CH₂)_(m)SO₃H.
 8. The compound of claim 1 which has a Stokes shift ofabout 30 nm to about 250 nm.
 9. The compound of claim 1 which has aStokes shift of about 50 nm to about 225 nm.
 10. The compound of claim 1which has a Stokes shift of about 70 nm to about 200 nm.
 11. A compoundof Formula I or II:

or a salt or conjugate thereof; wherein R¹ and R², when taken alone, areindependently H, halo, hydroxyl, amino, C₁₋₆ alkylamino, C₂₋₁₂dialkylamino, C₁₋₆perfluoroalkyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, SO₃H or COOH;R³ and R⁴, when taken alone, are independently (CH₂)_(m)SO₃H,(CH₂)_(n)COOH or optionally substituted C₁₋₆ alkyl, wherein m and n areindependently integers from 1 to 6; R⁵ and R⁶ are taken together withthe nitrogen atom to which they are attached to form an optionallysubstituted 5- or 6-membered azacyclic ring substituted, optionallycontaining an additional heteroatom selected from N, O and S as a ringmember; R⁷ and R⁸ are independently H, C₁₋₄ alkyl, or phenyl; X and Yare independently CR⁹ ₂, NR¹⁰, O or S; and each R⁹ and R¹⁰ isindependently C₁ ₋₄ alkyl.
 12. The compound of claim 11, wherein saidazacyclic ring is substituted with a substituent (CH₂)_(p)Z, where p isan integer from 0 to 6, and Z is SO₃H, COOH, OH, and NH₂.
 13. Thecompound of claim 11, wherein each of X and Y is CR⁹ ₂.
 14. The compoundof claim 11, wherein each of R⁷ and R⁸ is H.
 15. The compound of claim11, wherein each of R¹ and R² is SO₃H.
 16. The compound of claim 11,wherein at least one of R³ and R⁴ is C₁₋₆ alkyl substituted with asubstituent selected from the group consisting of halo, hydroxyl, C₁₋₆alkyl, or amino
 17. The compound of claim 11 which has a Stokes shift ofabout 30 nm to about 250 nm.
 18. The compound of claim 11 which has aStokes shift of about 50 nm to about 225 nm.
 19. The compound of claim11 which has a Stokes shift of about 70 nm to about 200 nm.
 20. Acompound of Formula III:

or a salt or conjugate thereof; wherein R¹ and R², when taken alone, areindependently H, halo, hydroxyl, amino, C₁₋₆ alkylamino, C₂₋₁₂dialkylamino, C₁₋₆ perfluoroalkyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, SO₃H orCOOH; R³ and R⁴, when taken alone, are independently (CH₂)_(m)SO₃H,(CH₂)_(n)COOH or optionally substituted C₁₋₆ alkyl, wherein m and n areindependently integers from 1 to 6; R⁵ and R⁶, when taken alone, areindependently H, C₁₋₆ carbonyl or optionally substituted C₁₋₆ alkyl; R⁷and R⁸ are independently H, C₁₋₄ alkyl, or phenyl; and each R⁹ isindependently C₁₋₄ alkyl.
 21. The compound of claim 20, wherein each R⁹is methyl.
 22. The compound of claim 20, wherein each of R⁷ and R⁸ is H.23. The compound of claim 20, wherein at least one of R⁵ and R⁶ is C₁₋₆alkyl substituted with a substituent selected from the group consistingof SO₃H, COOH, OH, and NH₂, maleimide, thiol, isocyanate,isothiocyanate, disulfide, alkynyl, azidoyl and trialkoxysilane.
 24. Thecompound of claim 20, wherein each of R¹ and R² is SO₃H.
 25. Thecompound of claim 20, wherein at least one of R³ and R⁴ is C₁₋₆ alkylsubstituted with a substituent selected from the group consisting ofhalo, hydroxyl, C₁₋₆ alkyl, or amino
 26. The compound of claim 20,wherein each of R³ and R⁴ is (CH₂)_(m)SO₃H.
 27. The compound of claim 20which has a Stokes shift of about 30 nm to about 250 nm.
 28. Thecompound of claim 20 which has a Stokes shift of about 50 nm to about225 nm.
 29. The compound of claim 20 which has a Stokes shift of about70 nm to about 200 nm.
 30. A compound of Formula III:

or a salt or conjugate thereof; wherein R¹ and R², when taken alone, areindependently H, halo, hydroxyl, amino, C₁₋₆ alkylamino, C₂₋₁₂dialkylamino, C₁₋₆perfluoroalkyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, SO₃H or COOH;R³ and R⁴, when taken alone, are independently (CH₂)_(m)SO₃H,(CH₂)_(n)COOH or optionally substituted C₁₋₆ alkyl, wherein m and n areindependently integers from 1 to 6; R⁵ and R⁶ are taken together withthe nitrogen atom to which they are attached to form an optionallysubstituted 5- or 6-membered azacyclic ring substituted, optionallycontaining an additional heteroatom selected from N, O and S as a ringmember; R⁷ and R⁸ are independently H, C₁₋₄ alkyl, or phenyl; and eachR⁹ is independently C₁₋₄ alkyl.
 31. The compound of claim 30, whereinsaid azacyclic ring is substituted with a substituent (CH₂)_(p)Z, wherep is an integer from 0 to 6, and Z is SO₃H, COOH, OH, and NH₂.
 32. Thecompound of claim 30, wherein each R⁹ is methyl.
 33. The compound ofclaim 30, wherein each of R⁷ and R⁸ is H.
 34. The compound of claim 30,wherein each of R¹ and R² is SO₃H.
 35. The compound of claim 30, whereineach of R³ and R⁴ is C₁₋₆ alkyl substituted with a substituent selectedfrom the group consisting of halo, hydroxyl, C₁₋₆ alkyl, or amino 36.The compound of claim 30 which has a Stokes shift of about 30 nm toabout 250 nm.
 37. The compound of claim 30 which has a Stokes shift ofabout 50 nm to about 225 nm.
 38. The compound of claim 30 which has aStokes shift of about 70 nm to about 200 nm.
 39. A compound selectedfrom:

or a salt or conjugate thereof.
 40. The compound of any one of claim 1,11, 20, 30, or 39 which is conjugated to a nucleotide, nucleoside,nucleic acid, oligonucleotide, deoxyoligonucleotide, DNA fragment, RNAfragment, or a derivatized variant of one of these; or an antibody,amino acid, peptide, or protein.
 41. The compound of any one of claim 1,11, 20, 30, or 39, wherein the compound has an excitation wavelengthbetween 400 and 900 nm.
 42. The compound of claim 41, wherein thecompound has an emission wavelength that is at least 70 nm greater thanthe excitation wavelength.
 43. A fluorescing molecular complexcomprising: a donor dye capable of absorbing light at a first wavelengthand emitting excitation energy in response; and an acceptor dye capableof absorbing the excitation energy emitted by the donor dye andfluorescing at a second wavelength in response; wherein either saiddonor dye or said acceptor dye has the structure of Formula III:

or a salt or conjugate thereof; wherein R¹ and R², when taken alone, areindependently H, halo, hydroxyl, amino, C₁₋₆ alkylamino, C₂₋₁₂dialkylamino, C₁₋₆ perfluoroalkyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, SO₃H orCOOH; R³ and R⁴, when taken alone, are independently (CH₂)_(m)SO₃H,(CH₂)_(n)COOH or optionally substituted C₁₋₆ alkyl, wherein m and n areindependently integers from 1 to 6; R⁵ and R⁶, when taken alone, areindependently H, C₁₋₆ carbonyl or optionally substituted C₁₋₆ alkyl; R⁷and R⁸ are independently H, C₁₋₄ alkyl, or phenyl; and each R⁹ isindependently C₁₋₄ alkyl.
 44. The donor dye or acceptor dye of claim 43which has a Stokes shift of about 30 nm to about 250 nm.
 45. The donordye or acceptor dye of claim 43 which has a Stokes shift of about 50 nmto about 225 nm.
 46. The donor dye or acceptor dye of claim 43 which hasa Stokes shift of about 70 nm to about 200 nm.
 47. The donor dye oracceptor dye of claim 43, wherein the compound has an excitationwavelength between 400 and 900 nm.
 48. The donor dye or acceptor dye ofclaim 47, wherein the compound has an emission wavelength that is atleast 70 nm greater than the excitation wavelength.
 49. A fluorescingmolecular complex comprising: a donor dye capable of absorbing light ata first wavelength and emitting excitation energy in response; and anacceptor dye capable of absorbing the excitation energy emitted by thedonor dye and fluorescing at a second wavelength in response; whereineither said donor dye or said acceptor dye has the structure of FormulaIII:

or a salt or conjugate thereof; wherein R¹ and R², when taken alone, areindependently H, halo, hydroxyl, amino, C₁₋₆ alkylamino, C₂₋₁₂dialkylamino, C₁₋₆perfluoroalkyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, SO₃H or COOH;R³ and R⁴, when taken alone, are independently (CH₂)_(m)SO₃H,(CH₂)_(n)COOH or optionally substituted C₁₋₆ alkyl, wherein m and n areindependently integers from 1 to 6; R⁵ and R⁶ are taken together withthe nitrogen atom to which they are attached to form an optionallysubstituted 5- or 6-membered azacyclic ring substituted, optionallycontaining an additional heteroatom selected from N, O and S as a ringmember; R⁷ and R⁸ are independently H, C₁₋₄ alkyl, or phenyl; and eachR⁹ is independently C₁₋₄ alkyl.
 50. The donor dye or acceptor dye ofclaim 49, wherein said azacyclic ring is substituted with a substituent(CH₂)_(p)Z, where p is an integer from 0 to 6, and Z is SO₃H, COOH, OH,and NH₂.
 51. The donor dye or acceptor dye of claim 49 which has aStokes shift of about 30 nm to about 250 nm.
 52. The donor dye oracceptor dye of claim 49 which has a Stokes shift of about 50 nm toabout 225 nm.
 53. The donor dye or acceptor dye of claim 49 which has aStokes shift of about 70 nm to about 200 nm.
 54. The donor dye oracceptor dye of claim 49, wherein the acceptor dye has an excitationwavelength between 400 and 900 nm.
 55. The donor dye or acceptor dye ofclaim 54, wherein the acceptor dye has an emission wavelength that is atleast 70 nm greater than the excitation wavelength.
 56. A substantiallypure composition of Formula I or II:

or a salt or conjugate thereof; wherein R¹ and R², when taken alone, areindependently H, halo, hydroxyl, amino, C₁₋₆ alkylamino, C₂₋₁₂dialkylamino, C₁₋₆ perfluoroalkyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, SO₃H orCOOH; R³ and R⁴, when taken alone, are independently (CH₂)_(m)SO₃H,(CH₂)_(n)COOH or optionally substituted C₁₋₆ alkyl, wherein m and n areindependently integers from 1 to 6; R⁵ and R⁶, when taken alone, areindependently H, C₁₋₆ carbonyl or optionally substituted C₁₋₆ alkyl; R⁷and R⁸ are independently H, C₁₋₄ alkyl, or phenyl; X and Y areindependently CR⁹ ₂, NR¹⁰, O or S; and each R⁹ and R¹⁹ is independentlyC₁₋₄ alkyl.
 57. The substantially pure composition of claim 56, whereineach of X and Y is CR⁹ ₂.
 58. The substantially pure composition ofclaim 56, wherein each of R⁷ and R⁸ is H.
 59. The substantially purecomposition of claim 56, wherein at least one of R⁵ and R⁶ is C₁₋₆ alkylsubstituted with a substituent selected from the group consisting ofSO₃H, COOH, OH, NH₂, maleimide, thiol, isocyanate, isothiocyanate,disulfide, alkynyl, azidoyl and trialkoxysilane.
 60. The substantiallypure composition of claim 56, wherein at least one of R³ and R⁴ is C₁₋₆alkyl substituted with a substituent selected from the group consistingof halo, hydroxyl, C₁₋₆ alkyl, or amino.
 61. The substantially purecomposition of claim 56, wherein each of R¹ and R² is SO₃H.
 62. Thesubstantially pure composition of claim 56, wherein each of R³ and R⁴ is(CH₂)_(m)SO₃H.
 63. The substantially pure composition of claim 56 whichhas a Stokes shift of about 30 nm to about 250 nm.
 64. The substantiallypure composition of claim 56 which has a Stokes shift of about 50 nm toabout 225 nm.
 65. The substantially pure composition of claim 56 whichhas a Stokes shift of about 70 nm to about 200 nm.
 66. A substantiallypure composition of Formula I or II:

or a salt or conjugate thereof; wherein R¹ and R², when taken alone, areindependently H, halo, hydroxyl, amino, C₁₋₆ alkylamino, C₂₋₁₂dialkylamino, C₁₋₆ perfluoroalkyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, SO₃H orCOOH; R³ and R⁴, when taken alone, are independently (CH₂)_(m)SO₃H,(CH₂)_(n)COOH or optionally substituted C₁₋₆ alkyl, wherein m and n areindependently integers from 1 to 6; R⁵ and R⁶ are taken together withthe nitrogen atom to which they are attached to form an optionallysubstituted 5- or 6-membered azacyclic ring substituted, optionallycontaining an additional heteroatom selected from N, O and S as a ringmember; R⁷ and R⁸ are independently H, C₁₋₄ alkyl, or phenyl; X and Yare independently CR⁹ ₂, Ne, O or S; and each R⁹ and R¹⁰ isindependently C₁₋₄ alkyl.
 67. The substantially pure composition ofclaim 66, wherein said azacyclic ring is substituted with a substituent(CH₂)_(p)Z, where p is an integer from 0 to 6, and Z is SO₃H, COOH, OH,and NH₂.
 68. The substantially pure composition of claim 66, whereineach of X and Y is CR⁹ ₂
 69. The substantially pure composition of claim66, wherein each of R⁷ and R⁸ is H.
 70. The substantially purecomposition of claim 66, wherein each of R¹ and R² is SO₃H.
 71. Thesubstantially pure composition of claim 66, wherein at least one of R³and R⁴ is C₁₋₆ alkyl substituted with a substituent selected from thegroup consisting of halo, hydroxyl, C₁₋₆ alkyl, or amino.
 72. Thesubstantially pure composition of claim 66 which has a Stokes shift ofabout 30 nm to about 250 nm.
 73. The substantially pure composition ofclaim 66 which has a Stokes shift of about 50 nm to about 225 nm. 74.The substantially pure composition of claim 66 which has a Stokes shiftof about 70 nm to about 200 nm.
 75. A substantially pure composition ofFormula III:

or a salt or conjugate thereof; wherein R¹ and R², when taken alone, areindependently H, halo, hydroxyl, amino, C₁₋₆ alkylamino, C₂₋₁₂dialkylamino, C₁₋₆ perfluoroalkyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, SO₃H orCOOH; R³ and R⁴, when taken alone, are independently (CH₂)_(m)SO₃H,(CH₂)_(n)COOH or optionally substituted C₁₋₆ alkyl, wherein m and n areindependently integers from 1 to 6; R⁵ and R⁶, when taken alone, areindependently H, C₁₋₆ carbonyl or optionally substituted C₁₋₆ alkyl; R⁷and R⁸ are independently H, C₁₋₄ alkyl, or phenyl; and each R⁹ isindependently C₁₋₄ alkyl.
 76. The substantially pure composition ofclaim 75, wherein each R⁹ is methyl.
 77. The substantially purecomposition of claim 75, wherein each of R⁷ and R⁸ is H.
 78. Thesubstantially pure composition of claim 75, wherein at least one of R⁵and R⁶ is C₁₋₆ alkyl substituted with a substituent selected from thegroup consisting of SO₃H, COOH, OH, and NH₂, maleimide, thiol,isocyanate, isothiocyanate, disulfide, alkynyl, azidoyl andtrialkoxysilane.
 79. The substantially pure composition of claim 75,wherein each of R¹ and R² is SO₃H.
 80. The substantially purecomposition of claim 75, wherein at least one of R³ and R⁴ is C₁₋₆ alkylsubstituted with a substituent selected from the group consisting ofhalo, hydroxyl, C₁₋₆ alkyl, or amino.
 81. The substantially purecomposition of claim 75, wherein each of R³ and R⁴ is (CH₂)_(m)SO₃H. 82.The substantially pure composition of claim 75 which has a Stokes shiftof about 30 nm to about 250 nm.
 83. The substantially pure compositionof claim 75 which has a Stokes shift of about 50 nm to about 225 nm. 84.The substantially pure composition of claim 75 which has a Stokes shiftof about 70 nm to about 200 nm.
 85. A substantially pure composition ofFormula III:

or a salt or conjugate thereof; wherein R¹ and R², when taken alone, areindependently H, halo, hydroxyl, amino, C₁₋₆ alkylamino, C₂₋₁₂dialkylamino, C₁₋₆perfluoroalkyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, SO₃H or COOH;R³ and R⁴, when taken alone, are independently (CH₂)_(m)SO₃H,(CH₂)_(n)COOH or optionally substituted C₁₋₆ alkyl, wherein m and n areindependently integers from 1 to 6; R⁵ and R⁶ are taken together withthe nitrogen atom to which they are attached to form an optionallysubstituted 5- or 6-membered azacyclic ring substituted, optionallycontaining an additional heteroatom selected from N, O and S as a ringmember; R⁷ and R⁸ are independently H, C₁₋₄ alkyl, or phenyl; and eachR⁹ is independently C₁₋₄ alkyl.
 86. The substantially pure compositionof claim 85, wherein said azacyclic ring is substituted with asubstituent (CH₂)_(p)Z, where p is an integer from 0 to 6, and Z isSO₃H, COOH, OH, and NH₂.
 87. The substantially pure composition of claim85, wherein each R⁹ is methyl.
 88. The substantially pure composition ofclaim 85, wherein each of R⁷ and R⁸ is H.
 89. The substantially purecomposition of claim 85, wherein each of R¹ and R² is SO₃H.
 90. Thesubstantially pure composition of claim 85, wherein each of R³ and R⁴ isC₁₋₆ alkyl substituted with a substituent selected from the groupconsisting of halo, hydroxyl, C₁₋₆ alkyl, or amino
 91. The substantiallypure composition of claim 85 which has a Stokes shift of about 30 nm toabout 250 nm.
 92. The substantially pure composition of claim 85 whichhas a Stokes shift of about 50 nm to about 225 nm.
 93. The substantiallypure composition of claim 85 which has a Stokes shift of about 70 nm toabout 200 nm.
 94. A process for preparing a compound of the formula:

said process comprising: a. dissolving a first compound having theformula

a second compound having the formula

and triethylamine into a mixture comprising dimethylformamide (DMF) andH₂O; and b. heating the mixture containing the first compound, thesecond compound, and triethylamine to between about 80° C. to about 85°C. for a period of about 2 hours.
 95. A process for preparing a compoundof the formula:

said process comprising: a. dissolving a first compound having theformula

β-alanine, and triethylamine into a mixture comprising dimethylformamide(DMF) and H₂O; and b. heating the mixture containing the first compound,β-alanine, and triethylamine to between about 80° C. to about 85° C. fora period of about 2 hours.